The present invention relates to a vaccine formulation for vaccinating equidae, in particular horses. It also relates to a corresponding vaccination method.
A relatively wide variety of horse pathologies exist. Apart from the well-known respiratory pathologies, such as rhinopneumonitis and influenza, horses are susceptible, in particular on the American continent, to various encephalomyelites. Finally, horses exhibit a variety of other pathologies among which tetanus, Lyme disease and equine arteritis may be mentioned, in particular, without forgetting the risks of exposure to the rabies virus.
The circumstances under which horses are exposed to various pathogenic microorganisms have been increased by the movement of large numbers of horses over substantial distances by land or by air, such that the risk of infection tends to increase.
However, in view of the high cost of these animals, in particular in the case of breeding animals, saddle horses and racehorses, it is economically important to control, as far as possible, the risks of infection, which translate into the animal being unavailable for long periods, if not actually being lost. A certain number of horse vaccines, whose efficacy varies, already exist.
Thus, inactivated or subunit vaccines, all of which, however exhibit some limitations expressed as incomplete or short-term protection and, possibly, safety problems linked to the adjuvants employed, have been developed for the equine rhinopneumonitis which is caused by the different strains of equine herpesvirus (EHV).
Attempts are also being made to use vaccination to prevent equine influenza, which is another important pathology. The vaccines employed are inactivated or subunit vaccines which, while they are effective to a certain degree, are nevertheless not without problems. Thus, protection is frequently not complete and is generally of a relatively brief duration, thereby requiring revaccinations, as in the case of rhinopneumonitis. Safety problems may also be encountered.
Vaccines based on antitetanus toxoid have also been developed and are undeniably effective.
Vaccines against encephalomyelites, some eastern encephalomyelites, western encephalomyelitis and Venezuelan encephalomyelitis, whose efficacy is still poorly known, also exist.
For reasons of economy, on the one hand, and the rational management of equine vaccinations on the other, multivalent vaccine formulations have already been proposed for preventing several of these infectious diseases.
The combinations which have so far been developed have been achieved using inactivated vaccines or live vaccines and, where appropriate, mixtures of these vaccines. Their implementation poses problems of compatibility between valencies and of stability. Thus, it is necessary to ensure compatibility between the different valencies, both with regard to the different antigens employed and with regard to the formulations themselves, in particular when inactivated vaccines and live vaccines are combined at the same time. There is also the problem of preserving such combined vaccines and also that of their safety, in particular in the presence of adjuvant. In general, these vaccines are relatively expensive.
Furthermore, these formulations have not enabled three of the main valencies, namely the equine influenza, rhinopneumonitis, in particular EHV-1 and EHV-4, and tetanus valencies, to be combined. For example, combinations of the influenza and encephalomyelitis valencies, or of the rhinopneumonitis and encephalomyelitis valencies, are known.
The Patent Applications WO-A-90 11 092, WO-A-93 19 183, WO-A-94 21 797 and WO-A-95 20 660 have proposed using the recently developed technique of polynucleotide vaccines. It is known that these vaccines use a plasmid which is capable of expressing, in the host cells, the antigen which is inserted into the plasmid. All the routes of administration have been proposed (intraperitoneal, intravenous, intramuscular, transcutaneous, intradermal, mucosal, etc.). Different means of vaccination may also be used, such as DNA deposited on the surface of gold particles and projected so as to penetrate into the skin of the animal (Tang et al., Nature 356, 152-154, 1992) and injections by means of a liquid jet, which makes it possible to transfect, at one and the same time, skin, muscle, fatty tissues and mammary tissues (Furth et al., Analytical Biochemistry, 205, 365-368, 1992) (See also U.S. Pat. Nos. 5,846,946, 5,620,896, 5,643,578, 5,580,589, 5,589,466, 5,693,622, and 5,703,055; Science, 259:1745-49, 1993; Robinson et al., seminars in IMMUNOLOGY, 9:271-83, 1997; Luke et al., J. Infect. Dis. 175(1):91-97, 1997; Norman et al., Vaccine, 15(8):801-803, 1997; Bourne et al., The Journal of Infectious Disease, 173:800-7, 1996; and, note that generally a plasmid for a vaccine or immunological composition can comprise DNA encoding an antigen operatively linked to regulatory sequences which control expression or expression and secretion of the antigen from a host cell, e.g., a mammalian cell; for instance, from upstream to downstream, DNA for a promoter, DNA for a eukaryotic leader peptide for secretion, DNA for the antigen, and DNA encoding a terminator.
The polynucleotide vaccines can use both naked DNAs and formulated DNAs, for example within liposomes or cationic lipids.
Polynucleotide vectors which integrate the HA or NT genes have been tried out in mice, ferrets and chickens in the case of the influenza virus. No data are available for the horse.
With regard to tetanus, it has recently been reported that immunization of mice with a plasmid which expresses the non-toxic C-terminal region of the tetanus toxin, together with the C fragment, induced the appearance of seroprotective antibodies in the mouse.
However, it is not possible to transpose directly the teaching of the results obtained in these animals of short lifespan to other mammals, in particular mammals of large size.
There is still a requirement, therefore, to improve the protection of equidae, in particular horses, against infectious pathologies.
The invention proposes to provide a multivalent vaccine formulation which makes it possible to vaccinate equidae, in particular horses, against a number of pathogenic agents.
Another object of the invention is to provide such a vaccine formulation which combines different valencies while at the same time exhibiting the requisite criteria of compatibility and stability of the valencies between themselves.
Another object of the invention is to provide such a vaccine formulation which makes it possible to combine different valencies in one and the same excipient.
Another object of the invention is to provide such a vaccine formulation which is easy to implement and inexpensive.
Yet another object of the invention is to provide such a formulation and a method of vaccinating horses which makes it possible to obtain a protection, including a multivalent protection, which is associated with a high level of efficacy and is of long duration while exhibiting a high degree of safety.
The present invention therefore relates to a vaccine formulation against pathologies of equidae, in particular horses, which comprises at least 3 polynucleotide vaccine valencies, each of which comprises an plasmid integrating so as to express it, in vivo in the cells, a gene of an equine pathogen valency, with these valencies being selected from the group consisting of equine rhinopneumonitis virus, EHV, equine influenza virus, EIV, and tetanus (Cl. tetani), with these plasmids comprising, for each valency, one or more of the genes selected from the group consisting of gB and gD in the case of the equine rhinopneumonitis virus, HA, NP and N in the case of the equine influenza virus, and a gene which encodes all or part of the C subunit of the tetanus toxin.
In the present invention, valency is understood as meaning at least one antigen which ensures protection against the virus of the pathogen under consideration, with the valency being able to contain, as a subvalency, one or more natural or modified genes of one or more strains of the pathogen under consideration.
Pathogenic agent gene is understood as meaning not only the complete gene but also the different nucleotide sequences, including fragments, which retain the ability to induce a protective response. The gene concept covers the nucleotide sequences which are equivalent to those described precisely in the examples, that is to say the sequences which are different but which encode the same protein. It also covers the nucleotide sequences of other strains of the pathogen under consideration which ensure crossprotection or a protection which is specific for a strain or a group of strains. It also covers the nucleotide sequences which have been modified in order to facilitate expression in vivo by the animal host but which encode the same protein.
Thus, particularly preferably, the vaccine according to the invention comprises, in the equine rhinopneumonitis valency, at least one antigen from the EHV-1 strain and at least one antigen from the EHV-4 strain, with these antigens preferably being the same type of antigen.
The therapeutically effective quantities of the polynucleotide valencies are contained, or are intended to be contained, in an excipient which is suitable for administering to the animal, preferably for intramuscular administration. Preferably, this excipient is an aqueous excipient which lacks oily constituents.
With regard to the equine rhinopneumonitis valency, preference is given to combining the gB and gD genes, preferably from EHV strains, in particular the 1 and 4 strains.
With regard to the equine influenza valency, preference is given to using the gene which encodes the haemagglutinin HA or to using the combination of the genes which encode HA and NP. The influenza virus HA sequences, in particular from the different strains encountered in the territory, are preferably combined in one and the same vaccine. On the other hand, NP ensures crossprotection and it is possible, therefore, to be contented with the sequence from one single strain of the virus.
In the case of the tetanus valency, preference is given to the C subunit, where appropriate modified by mutation or deletion.
Combining genes which encode several antigens of one and the same valency or of one and the same strain in a valency can be effected by mixing plasmids which express a single antigen or, on the contrary, by inserting several genes into one and the same plasmid.
While combining the different valencies of the vaccine according to the invention can preferably be effected by mixing polynucleotide plasmids which express one or more antigens of each valency, it is also possible to envisage having several antigens of several valencies being expressed by one and the same vector of the plasmid type.
In an improved form of the invention, the formulation can also include one or more other valencies of other equine pathogens, in particular valencies of the eastern encephalomyelitis virus, EEV, of the western encephalomyelitis virus, WEV, and of the Venezuelan encephalomyelitis virus, VEV, preferably all three simultaneously.
These valencies can also advantageously include the valency of Lyme disease, B. burgdorferi, of equine arteritis (EAV) and of rabies.
The genes of the abovementioned encephalomyelites which are used are the genes for the C and E2 antigens, preferably the E2 gene on its own or the combination of the two genes E2 and C.
In the case of the Lyme disease valency, a selection is made between the OspA, OspB and p100 genes, with OspA being preferred.
In the case of equine arteritis, the E, M and N genes, which are used either on their own or in combination are selected.
In the case of rabies, the G gene is selected.
A vaccine formulation according to the invention can be presented in a dose volume of between 0.1 and 10 ml, in particular of between 1 and 5 ml.
The dose is generally between 10 ng and 1 mg, in particular between 100 ng and 500 xcexcg, preferably between 1 xcexcg and 250 xcexcg per plasmid type.
Preference is given to using naked plasmids, which are simply placed in the vaccination excipient, which is in general physiological saline (0.9% NaCl), ultrapure water, TE buffer, etc. It is, of course, possible to use all the polynucleotide vaccine formulations described in the prior art.
Each plasmid comprises a promoter which is capable of ensuring expression of the inserted gene under its control in the host cells. In general, the promoter is a strong eukaryotic promoter, in particular an early promoter of the cytomegalovirus CMV-IE of human or murine origin, or else, where appropriate, of another origin such as rat, pig or guinea pig.
More generally, the promoter can be either of viral origin or of cellular origin. Viral promoters other than the CMV-IE promoter which may be mentioned are the early or later promoters of the SV 40 virus or the LTR promoter of the Rous sarcoma virus. The promoter can also be a promoter of the virus from which the gene is derived, for example the gene""s own promoter.
A cellular promoter which may be mentioned is the promoter of a gene of the cytoskeleton, for example the desmin promoter (Polmont et al., Journal of Sub-microscopic Cytology and Pathology, 1990, 22, 117-122; and Zhenlin et al., Gene, 1989, 78, 243-254), or else the actin promoter.
When several genes are present in one and the same plasmid, they may be presented within the same transcription unit or within two different units.
The invention also relates to monovalent vaccine formulations which comprise one or more plasmids which encode one or more genes of one of the abovementioned pathogenic agents, in particular of rhinopneumonitis or of Lyme disease, of equine arteritis, of eastern encephalomyelitis, of western encephalomyelitis and of Venezuelan encephalomyelitis, with the genes being those described above. These formulations can comprise the above-mentioned features as regards the choice of the genes from one and the same pathogen and their combination, the composition of the plasmids, the dose volumes, the dosages, etc.
The present invention also relates to a method of vaccinating equidae, in particular horses, against infectious diseases, which method comprises administering an effective dose of a multivalent or monovalent vaccine formulation such as described above.
This method of vaccination comprises administering one or more doses of the vaccine formulation.
The vaccine formulations according to the invention can be administered by the different routes of is administration which have been proposed within the general context of polynucleotide vaccination and using known administration techniques. However, the intramuscular route is distinctly preferred.
It is also possible to vaccinate by the intradermal route by means of a liquid jet, preferably by means of multiple jets, with the aid of an injector, in particular an injector which uses an injection head which is fitted with several holes or nozzles, in particular, from 5 to 6 holes or nozzles, such as the Pigjet appliance, which is produced and distributed by the firm Endoscoptic, Laons, France.
The dose volume in the case of such an appliance is preferably reduced to between 0.1 and 0.9 ml, in particular between 0.2 and 0.6 ml, and advantageously between 0.4 and 0.5 ml, with it being possible for the volume to be administered in one or more, preferably 2, applications.
The abovementioned monovalent vaccines can be used, in particular, for preparing the polyvalent vaccine according to the invention.
The monovalent vaccine formulations can also be used in combination with a vaccine of another type (whole live or inactivated, recombinant or subunit) against another pathology or as a booster for a vaccine as described below.
Thus, the present invention also relates to the use of one or more plasmids according to the invention for producing a vaccine which is intended for vaccinating horses which have been initially vaccinated with a conventional first vaccine (monovalent or multivalent) which is of the same type as those of the prior art and which is selected, in particular, from the group consisting of whole live vaccine, whole inactivated vaccine, subunit vaccine or recombinant vaccine, with this first vaccine exhibiting (that is to say containing or being able to express) the antigen or the antigens encoded by the plasmid or the plasmids or (an) antigen(s) which ensure(s) crossprotection.
Remarkably, the polynucleotide vaccine has a powerful booster effect which translates into an amplification of the immune response and the establishment of long-term immunity.
In general, the first-vaccination vaccines can be selected from the commercial vaccines which can be obtained from the different veterinary vaccine producers.
In one preferred embodiment of the process according to the invention, an effective dose of the conventional vaccine, in particular an inactivated, live, attenuated or recombinant vaccine, or else a subunit vaccine, is firstly administered to the animal so as to ensure an initial vaccination and the polyvalent or monovalent vaccine according to the invention is administered after a waiting period of preferably from 2 to 4 weeks.
The invention also relates to a vaccination kit which combines a vaccine formulation according to the invention and a first-vaccination vaccine such as described above. It also relates to a vaccine formulation according to the invention which is accompanied by a leaflet which indicates the use of this formulation as a booster for a first vaccination such as described above.
The invention also relates to the method for preparing the vaccination formulations, namely the preparation of the valencies and their mixtures, as is evident from this description.